WO2004047870A1 - Conjugats de nanoparticules et leur procede de production - Google Patents

Conjugats de nanoparticules et leur procede de production Download PDF

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WO2004047870A1
WO2004047870A1 PCT/GB2003/005157 GB0305157W WO2004047870A1 WO 2004047870 A1 WO2004047870 A1 WO 2004047870A1 GB 0305157 W GB0305157 W GB 0305157W WO 2004047870 A1 WO2004047870 A1 WO 2004047870A1
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nanoparticle
molecules
hydrophilic polymer
nanoparticles
flexible hydrophilic
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PCT/GB2003/005157
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English (en)
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Robert Wilson
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The University Of Liverpool
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Priority to US10/537,164 priority Critical patent/US20060148124A1/en
Priority to CA002507687A priority patent/CA2507687A1/fr
Priority to AU2003285531A priority patent/AU2003285531A1/en
Priority to EP03778528A priority patent/EP1565216A1/fr
Priority to JP2004554698A priority patent/JP2006513175A/ja
Publication of WO2004047870A1 publication Critical patent/WO2004047870A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6883—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb

Definitions

  • the present invention relates to nanoparticle conjugates, in particular those which are useful in biomolecular assays, and to methods for their production.
  • conjugates are believed to have potential as novel intravascular probes for both sensing (e.g., imaging) and therapeutic purposes (e.g., drug delivery) (Proc. Natl. Acad. Sci. USA, 99 (2002) 12617-12621).
  • the requirements of these conjugates are in many respects similar to those used in biomolecular assays, but a further requirement is that the conjugate should be biocompatible and, for in vivo applications, should be biodegradable or able to pass through the biological particulate filter known as the reticuloendothelial system.
  • proteins contain chemical groups (-NH 2 , -SH 2 , etc.) that bind strongly to certain types of nanoparticle, but if necessary the number of such ⁇ groups can be increased with, for example, a thiolating reagent such as 2- iminothiolane, or by genetic engineering. Fine details of the process by which proteins become non-specifically bound to nanoparticles are not well understood, but it has been postulated that a series of electrostatic and chemical interactions accompanied by conformation changes is involved. The most widespread use of this method is for conjugating antibodies to gold nanoparticles (J. Histochem. Cytochem., 36 (1988) 401-407 and Biotechnic & Histochem., 75 (2002) 203-242).
  • a small excess of antibodies are incubated with the nanoparticles for a short time under alkaline conditions. Then unbound protein is removed and the conjugate is stabilized with a blocking agent such as polyethylene glycol.
  • a blocking agent such as polyethylene glycol.
  • the optimal amount of protein required for conjugation can be determined by means of a flocculation assay. When electrolytes are added to incompletely conjugated particles they flocculate. The flocculation of gold nanoparticles can be monitored by the decrease and/or red shift of the plasmon absorption band at about 520 nm. Non- specifically conjugated proteins stabilize the nanoparticles by mutual repulsion. The minimum amount of protein needed to prevent flocculation is determined by titration and often corresponds to a single monolayer bound to the surface of the nanoparticles.
  • Monovalent methods of conjugation involve incubating the nanoparticles with an excess of molecules that comprise a chemical group that binds to the particles and a binding site that can participate in biomolecular or other applications (as shown in Figure 1). Unbound molecules are removed in a subsequent purification step such as ultra-centrifugation or gel exclusion chromatography. Monovalent conjugation may also be the first step in a more complicated protocol.
  • a recent conference report described the conjugation of long chain mercaptoalcohols to gold nanoparticles (Presented by V.H. Perez-Luna at the Nanotechnology in bioengineering: applications to detection, diagnostics and sensing conference, 7 th Nov, 2002, Indiana, USA).
  • Monovalent conjugates are characterized by a single dissociation constant K- d which depends on the affinity of individual conjugation substituents for the nanoparticle, but multivalent conjugates are characterized by a series of dissociation constants (combined in K t ).
  • K- d dissociation constant
  • K t dissociation constants
  • the latter compound was originally synthesized for multivalent attachment of peptides to macroscopic gold substrates (Science, 261 (1993) 73-76). It remained bound to these substrates even when it was heated to 180°C for 7 days.
  • a conjugation substituent bound to a nanoparticle enhances the binding of a second conjugation substituent to the same nanoparticle depends on the distance by which the conjugation substituents are separated, and the amount of steric hindrance exerted by the chemical structure that links them together.
  • Dextrans are flexible polymers of glucose that are known to resist nonspecific adsorption and allow fast kinetics in biomolecular assays (Sensor Actuat. B-Chem., 5 (1991) 78-84; Biomaterials, 21 (2000) 957-966). The same flexibility that favours fast kinetics also reduces the amount of steric hindrance between conjugation substituents in the same multivalent molecule.
  • aminodextran (amdex) products can form multivalent conjugates with metal and semiconductor nanoparticles (see U.S. Pat. Nos. 5,248,772; 5,552,086 and 5,945,293 z ⁇ Langmuir, 16 (2000) 3107-3118).
  • Aminodextrans have been used to conjugate biochemically active molecules such as ouabain to gold nanoparticles (Eur. J. Cell Biol. 45 (1987) 200-208; Invest. Opthalmol. & Vis. Sci., 26 (1985) 1002-1013; J. Cell Biol, 105 (1987) 2589- 2601; J. Bacteriol, 169 (1987) 3531-3538).
  • Nanoparticle conjugate dissociation can also be avoided by entrapping the particles in a shell from which they are unable to escape (Figure 5).
  • Metallic and semiconductor nanoparticles can be entrapped in a shell formed by polymerization of mercaptopropyltrimethoxy silane (Langmuir, 13 (1997) 3921- 3926; Chem. Mater. 14 (2002) 2113-2119).
  • the shell confers stability on the nanoparticles and further reaction with other silanes introduces functional groups that can be covalently attached to binding molecules such as biotin (Chem. Mater. 14 (2002) 2113-2119).
  • Nanoparticles can also be entrapped by cross-linking dextran with epichlorohydrin (Bioconjugate.
  • Nanoparticle conjugates prepared in this way have been used for non-invasive magnetic resonance imaging and cancer therapy. When nanoparticles are exposed to a succession of polyelectrolyte solutions of alternating charge they become entrapped in a polymer shell that is robust enough to remain intact when core particle is dissolved. Gold nanoparticles coated with an anionic layer of monovalent mercapto compounds have subsequently been entrapped in alternate layers of sodium poly(styrenesulfonate) and poly(diallyldimethylammonium) chloride (J. Phys.
  • Biomolecular binding assays can be divided into two categories depending on whether the concentration of the labeled reagent is 1) higher than the concentration of the analyte or 2) about the same as or less than the concentration of the analyte.
  • the former (1) are known as regent excess assays and the latter (2) are known as reagent limited assays.
  • a sandwich immunoassay is an example of a regent excess assay and a competitive immunoassay is an example of a reagent limited assay.
  • the importance of this distinction between reagent excess and reagent limited assays has been recognized for many years. It is known to determine what factors will ultimately limit the sensitivity of an assay (Principles and Practice of Immunoassay, 2nd ed.
  • K a is known as the intrinsic affinity of the probe molecule
  • [PA] is the concentration of the probe molecule bound to the analyte
  • [P] is the free probe molecule concentration
  • [A] is the free analyte concentration.
  • K a is equal to the ratio of the association and dissociation rate constants: Any factor that increases the association rate constant (k a ) or decreases the dissociation rate constant (k d ) will increase the intrinsic affinity (K a ) of the probe molecule for the analyte.
  • nanoparticle labels are interesting is because they provide a way to increase k a and decrease k d . Before discussing how this can be done it is necessary to define another value known as the functional affinity.
  • Equations I and II describe the equilibrium between a monovalent probe molecule and the analyte, but many probe molecules have more than one binding site.
  • the antibodies most often used in immunoassays, for example, are bivalent, which means that in some cases one antibody may be bound to the same analyte molecule by two binding sites. When this happens the reaction at equilibrium is described by the equation:
  • K f is known as the functional affinity of the probe molecule, and the numerical subscripts indicate the number of bonds between the probe molecule and the analyte.
  • the value of K f may be orders of magnitude greater than the value of K a indicating that the probe molecule is more tightly bound to the analyte.
  • the increase in K f is due to a decrease in the value of k d .
  • FIG 7 shows two nanoparticle conjugates (NPCs) in solution with a molecule of the corresponding analyte.
  • Conjugate A has many identical probe molecules conjugated to the same nanoparticle and conjugate B has only one probe molecule of the same type conjugated-to.a nanoparticle.
  • Conjugate A has a much higher probability of binding to the analyte X because most of the surface of conjugate B does not have any binding sites.
  • the value of k a for conjugate A is •higher than for conjugate B and therefore the value of K f is also higher.
  • Figure 8 shows the same nanoparticle conjugates A and B bound to the corresponding analyte.
  • one molecule of the analyte X can accommodate more than one binding site and therefore conjugate A, but not conjugate B, can participate in polyvalent binding.
  • Polyvalent binding decreases the value of k d , as explained above, and therefore once again conjugate A has a higher value of K f than conjugate B.
  • reagent limited assays The situation in reagent limited assays is more complicated because the .sensitivity may be influenced by the density of binding sites on the separation phase as well as the number of probe molecules conjugated to each nanoparticle.
  • reagent limited immunoassays for TNT Wilson et al. found that the sensitivity could be increased by using monovalent Fab fragments instead of bivalent whole antibodies (Anal Chem. 75 (2003) 4244-4249).
  • the sensitivity increased because the Fab fragments had a similar functional affinity for the separation phase as the analyte.
  • Bivalent whole antibodies had a higher functional affinity for the separation phase (a high affinity haptenylated dextran surface) than the analyte, and therefore high concentrations of analyte were required to produce a measurable change in the number of bivalent antibodies bound to the separation phase.
  • nanoparticles conjugated to a plurality of probe molecules When nanoparticles conjugated to a plurality of probe molecules are used, the potential for polyvalent binding to the separation phase, accompanied by a decrease in sensitivity, is even greater than when bivalent antibodies are used.
  • a further complication when considering reagent limited assays is that there are two possible formats based on 1) probe molecules conjugated to nanoparticles and 2) analyte analogues conjugated to nanoparticles.
  • FIG. 9A and B shows the situation when nanoparticles are conjugated to A) high and B) low numbers of probe molecules.
  • the value of k a for the binding of the conjugate to the analyte and the separation phase both increase as the numbers of probe molecules increase: .
  • This increase is accompanied by an increase in sensitivity provided polyvalent binding to the separation phase does not occur.
  • polyvalent binding to the separation phase does occur, high concentrations of analyte are required to produce a measurable change in the amount of conjugate bound to the separation phase, and sensitivity is low. This is what Goldman et al.
  • k a The value of k a is unaffected by an increase in the number of analyte analogues conjugated to each particle and therefore there is no increase in sensitivity.
  • Nanoparticles conjugated to high numbers of analyte molecules can participate in polyvalent binding to the separation phase. Maximum sensitivity in this type of reagent limited immunoassay is attained when low numbers of analyte analogues are conjugated to each nanoparticles.
  • a particular advantage of the hand-in-glove method described here is that it allows the number of probe molecules to be .determined prior to conjugation.
  • the probe molecules are linked to a high molecular weight dextran, which is then conjugated to the particles by a process of self-assembly.
  • the number of probe molecules conjugated to each particle is determined by the relationship between the size of the dextran and the diameter of the nanoparticles.
  • nanoparticles and biological molecules also makes them suitable for in vivo applications.
  • the requirements of these nanoparticle conjugates are in many respects similar to those used in biomolecular assays, except that the analytical probe molecules are replaced with other functional molecules.
  • Superparamagnetic metal oxide nanoparticles entrapped in a layer of dextran and conjugated to a membrane translocating peptide can be internalised by living cells. The cells can be detected by NMR imaging and could be retained on magnetic separation columns (Bioconjugate. Chem. 10 (1999) 186- 191).
  • nanoparticle conjugate for use in biomolecular assay, and in other applications, for which the number of functional molecules conjugated to the nanoparticles could be determined by more straightforward analytical techniques than has hitherto been the case. It would also be desirable to provide a nanoparticle conjugate which could be determined with greater sensitivity and/or reliability than has been known until now. It would be a further advantage to provide a nanoparticle conjugate which was relatively stable, preferably highly stable, and not readily dissociated. It would also be advantageous to provide a method for producing a nanoparticle conjugate for use in biomolecular assays or other applications in which the numbers and types of molecules conjugated to the particle could be more accurately controlled than has hitherto been the case. It would also be desirable if such a method for producing the nanoparticle conjugates allowed the number and type of molecules conjugated to the particle to be determined more accurately that has been known until now.
  • nanoparticle conjugates It is an object of the present invention to provide such nanoparticle conjugates and methods for making them.
  • One particular object of the invention is to provide nanoparticle conjugates that, when used in a biomolecular assay, are able to resist non-specific binding, will allow fast kinetics, and/or will resist displacement and exchange reactions.
  • Another object of the invention is to provide nanoparticle conjugates with controlled functional affinity.
  • Yet another object of the invention is to provide a nanoparticle conjugate comprising a controlled number of binding molecules for biomolecular assay applications.
  • the invention provides a method for the preparation of ⁇ nanoparticle conjugates comprising: a) providing a first reagent comprising a flexible hydrophilic polymer; b) providing a second reagent comprising at least one functional molecule capable of being substituted into the flexible hydrophilic polymer; c) providing a third reagent comprising nanoparticles; d) contacting the first reagent with the second reagent for a period of time and under conditions effective to allow substitution of the at least one functional molecule into the flexible hydrophilic polymer; e) before, during and/or after step d) providing the flexible hydrophilic polymer with a plurality of conjugation substituents capable, optionally after deprotection, of binding to the nanoparticles to provide an intermediate product comprising the flexible hydrophilic polymer substituted with the at least one functional molecule and a plurality of conjugation substituents capable, optionally after deprotection, of binding to the nanoparticles; f) if necessary, deprotecting
  • step g controlling, by means of suitable selection of reagents and reaction conditions, the number of intermediate product molecules binding to the nanoparticles in step g).
  • the method of the invention allows the preparation of nanoparticle conjugates wherein the number of functional molecules thereon can be controlled according to the end use of the nanoparticle conjugate.
  • the target end product of the method of the invention is a nanoparticle conjugate having a desired number of functional molecules per nanoparticle and the method of the invention permits selection of conditions which yield the desired target end product. If the desired number of functional molecules per nanoparticle is small then the degree of control permitted by the method of the invention is close, even allowing the operator of the method to reliably ensure that, for example, in a sample of nanoparticle conjugates obtained by the method of the invention, the mean number of functional molecules conjugated t ⁇ ' each nanoparticle is one.
  • the functional molecule may be an assay molecul and the mean number of such assay molecules per nanoparticle conjugate is controlled (probably to a relatively small number) with this end use in mind.
  • the functional molecule is intended for use in certain other assay applications or for use in drug delivery, for example, the mean number of such assay, drug delivery or drug molecules per nanoparticle conjugate is controlled (probably to a relatively large number) with this end use in mind.
  • the degree of control under these circumstances is less close but is still significant in that the operator is able to ensure, for example, that a large mean number of functional molecules conjugate to the nanoparticle.
  • the functional molecules may be directly functional (as assay molecules for example) or their functionality may be indirect in that they may be capable of binding further to other functional molecules.
  • Control of the mean number of functional molecules per nanoparticle in a given sample is preferably achieved by at least one of:
  • step d) selecting the relative concentrations of the first and second reagents in step d) to control the number of functional molecules substituted into each molecule of flexible hydrophilic polymer
  • the method of the invention may comprise determining at least approximately the desired mean number of, if necessary deprotected, intermediate product molecules to be bound to each nanoparticle in step g) and selecting the relative size of the flexible hydrophilic polymer and the nanoparticle such that the mean number of, if necessary deprotected, intermediate product molecules which can be accommodated on the surface of each nanoparticle at least approximately matches the desired number.
  • the desired number is small (for example less than about 10, less than about 5, from about 1 to 3 or only 1), the number of, if necessary deprotected, intermediate product molecules which can be accommodated on the surface of each nanoparticle at least almost exactly matches the desired number.
  • the method of the invention may also comprise determining at least approximately the desired number of functional molecules to be substituted into each molecule of flexible hydrophilic polymer in step d) and selecting accordingly the reagent concentrations and reaction conditions in step d).
  • the method of the invention may also comprise determining at least approximately the desired number of substituent molecules capable, optionally after deprotection, of binding to the nanoparticles to be substituted to each molecule of flexible hydrophilic polymer in step e) and selecting accordingly the reagent concentrations and reaction condition's in step e).
  • the relative sizes of the flexible hydrophilic polymer and the nanoparticle are selected to be effective to allow binding in step g) of a controlled number of the, if necessary deprotected, intermediate product molecules with the nanoparticles.
  • Also provided in accordance with the invention is a method for the preparation of a nanoparticle conjugate comprising: i. providing a first reagent comprising a flexible hydrophilic polymer having a plurality of at least one type of conjugation substituent capable, optionally after deprotection, of binding to a nanoparticle; ii. providing a second reagent comprising at least one functional molecule suitable for binding to target molecules, optionally in a biomolecular assay, and capable of being substituted into the flexible hydrophilic polymer; iii. providing a third reagent comprising nanoparticles capable of binding to the conjugation substituents of the flexible hydrophilic polymer; iv.
  • the method of the invention allows the production of a nanoparticle conjugate comprising a nanoparticle and a flexible hydrophilic polymer bound to the nanoparticle, the flexible hydrophilic polymer (and hence the nanoparticle) being substituted with a known mean number of functional molecules per nanoparticle conjugate.
  • the invention provides a method for conjugating one or more functional molecules to a nanoparticle. More specifically the invention in some cases concerns a method for substituting known mean numbers of one or more functional molecules into a flexible hydrophilic polymer that is also substituted with conjugation substitutents having chemical groups that are capable of binding to a nanoparticle, and a method for conjugating a known number of substituted polymer molecules to a nanoparticle.
  • the invention accordingly provides a method for producing nanoparticle conjugates having a number of functional molecules that is at least approximately known even without analytical determination of the nanoparticle conjugate after it has been prepared.
  • the invention also provides a method fpj producing a nanoparticle conjugate comprising:
  • the flexible hydrophilic polymer is selected with regard to the variable y to provide a nanoparticle conjugate having an at least approximately predetermined number of flexible hydrophilic polymer molecules per nanoparticle.
  • nanoparticle-based biomolecular assays are partly dependent on their functional affinity (number and affinity of assay binding sites per nanoparticle) and an aspect of this invention is to provide a method for exercising control over the functional affinity of nanoparticle conjugates.
  • the plurality of substituents capable of binding to a nanoparticle comprise mercapto (-SH) groups or 'alternatively either disulphide (-S-S-) or thioester (-COS-) groups that can be deprotected to provide ultimately the same conjugation as would be derived from mercapto groups.
  • the invention provides a nanoparticle conjugate comprising a nanoparticle conjugated to a functionalised flexible hydiOphilic polymer via a plurality of mercapto groups.
  • the invention also provides methods for producing such nanoparticles, as hereinbefore described. In effect the method of the invention enables the synthesis of the entire surface of the nanoparticle conjugate before its formation by conjugation. This has the significant advantage that if the surface is constructed at high concentration, the number of functional molecules substituted into the surface (before conjugation to the surface of the nanoparticle) can be determined directly, and any purification steps can be carried out using conventional techniques that give a high yield.
  • the flexible hydrophilic polymer is preferably selected from polysaccharides, polyethylene glycols, polyv n ⁇ l alcohols, polyacrylic acids, polyacrylamides, polyamides (including polyamino acids), polycarboxylated polymers (including polyaminoacids) and pseuodop lyamino acids.
  • suitable polysaccharides include natural polysaccharides such as dextran, fucoidan, arabinogalactan, chondroitin and its sulfates, dermatan, heparin, heparitin, hyaluronic acid, keratan, polygalacturonic acid, polyglucoronic acid, polymannuronic acid, inulin, polylactose, polyactosamine, polyinosinic acid, polysucrose, amylose, amylopectin, glycogen, glucan, nigeran, pullulan, irisin, asparagosin, sinistrin, tricitin, critesin, graminin, sitosin, lichenin, isolichenan, galactan, galactocaolose, luteose, mannans, mannocarolose, pustulan, laminarin, xanthene, xylan and copolymers, araboxylan, arabinogalactan
  • the plurality of conjugation substituents capable, optionally after deprotection, of binding to nanoparticles may effect such binding chemically, electrostatically, hydrophobically or by a combination thereof.
  • the flexible hydrophilic polymer is preferably provided with pendant substituents with such capability.
  • Such substituents preferably comprise a conjugation group (for conjugation to the nanoparticle) selected from sulphides (-S-), asymmetrical or symmetrical disulphides (-S-S-), selenides (-Sg-), diselenides (-Se-Se-), mercapto (thiol, sulphydryl, -SH), nitrile (-CN), isonitrile, nitro (-NO ), amino (NH 2 ), selenol (-SeH), trivalent phosphorous compounds, isothiocyanate, xanthate, thiocarbamate, phosphine, thioacid (-COSH) or dithioacid (-CSSH) and thioester (-COS-).
  • Particularly preferred conjugation group in this respect include mercapto (thiol, sulphydryl, -SH) and disulphide (-S-S-).
  • the number, per molecule of flexible hydrophilic polymer, of substituents capable, optionally after deprotection, of binding to the nanoparticles is greater than one, preferably greater than two, more preferably greater than about three, still more preferably greater than about five, most preferably greater than about ten.
  • the functional molecules may, for example, find applications in biomolecular assays, as ligands for targeting biochemical receptors, or as therapeutic or pharmacological agents.
  • suitable functional molecules include chelating agents, antigens, haptens, natural or synthetic peptides, natural or synthetic proteins, protein A, protein G, biotin, avidin, streptavidin, antibodies including monoclonal and polyclonal antibodies, Fab' fragments, Fab fragments, enzymes enzyme cofactors, hormones including steroid, amino acid, peptide and protein hormones, specific carbohydrates, natural and synthetic mono-, oligo- and polysaccharides, gene probes, natural and synthetic polynucleotides and oligonucleotides, lectins, growth factors, vitamins, drugs, hormones, receptor molecules, chimaeric or fusion molecules derived from two or more of these molecules.
  • the nanoparticle may comprise a metal, for example Au, Ag or a bimetallic composite thereof.
  • suitable nanoparticle materials include semiconductors such as the sulphides and selenides of Zn, Cd, Pb, Sn, Hg, Al, Ga, In, Ti, Si, Ag, Fe, Fe, Ni and Ca.
  • Preferred semiconductor nanoparticles include CdSe, ZnSe, CdTe, InP, InAs, PbSe, PbS and CdS.
  • Metal oxide nanoparticles such as iron oxide may also be used.
  • the nanoparticle may have a core-shell structure, in which case the shell may be a metal, semiconductor or metal oxide, and the core may be a metal, semiconductor, metal oxide or metalloid oxide. In some cases the core material may be chosen to render the nanoparticle conjugates responsive to a magnetic field.
  • Figure 1 illustrates a schematic representation of a monovalent conjugation method in which oligonucleotides with a terminal mercapto group are conjugated to gold nanoparticles
  • Figure 2 illustrates a comparison between the dissociation of monovalent and multivalent conjugates
  • Figure 3 illustrates a schematic representation of a simple multivalent (divalent) conjugation method based on dihydrolipoic acid (J. Am. Chem. Soc, 122 (2000) 12142-12150). Following conjugation the COOH groups of the conjugate are covalently linked to a protein molecule (avidin);
  • Figure 4 illustrates the structural formula of aminotrithiolate that has been used for the multivalent (trivalent) conjugation of antibodies to metallic nanoparticles.
  • This multivalent molecule binds so tightly to silver nanoparticles that aminodextrans, multivalently conjugated to the nanoparticles by a plurality of primary amine groups, are displaced by it (U:S, Pat No. 5,945,293 (1999));
  • Figure 5 illustrates the schematic representation of an entrapment method of conjugation based on mercaptopropyltrimethoxy silane.
  • step A the mercapto ⁇ groups bind to the nanoparticle and the alkoxy groups point outwards where they are available for cross-linking to each other and to other silanes in step B.
  • the other silanes have primary amine groups that can be covalently linked to binding molecules such as antibodies in step C;
  • Figure 6 illustrates that a nanoparticle conjugate has a greater probability of undergoing a collision with an analyte X than a nanoparticle conjugate B because most of the latter' s surface is unreactive. Therefore nanoparticle conjugate A has a higher k a , and hence a higher functional affinity, than nanoparticle B;
  • Figure 7 illustrates that nanoparticle conjugate A is bound to the analyte X at more than site, but nanoparticle conjugate B is only bound at one site. Therefore nanoparticle A has a lower k d , and hence higher functional affinity, than nanoparticle B;
  • Figure 8 illustrates that nanoparticle conjugate A has a higher probability of binding to the analyte X and the separation phase than NPC B because most of latter' s surface is unreactive. Therefore NPC A has a higher k a value for the analyte and the separation phase than NPC B.
  • NPC A may also be able to bind to more than one site on the separation phase, but NPC can only bind to one site. Therefore NPC A may also have a lower value of K d than NPC B;
  • Figure 9 illustrates that NPC a has a higher probability of binding to the separation phase than NPC B.
  • NPC A may also be able to bind to more than one site on the separation phase, but NPC B can only bind to one site.
  • NPC A has a higher functional affinity for the separation phase than for the analyte, but NPC B has a similar functional affinity for the separation phase ⁇ and the analyte.
  • Figure 10 is a schematic representation of the method used to conjugate PDP dextran linked probe molecules to NPs. Key: 1) the probe molecule (an ohgonucleotide in this example) is activated; 2) the probe molecule and PDP are linked to dextran; 3) the dextran is conjugated to the NP by a plurality of dative covalent bonds;
  • Figure 11 is a graphical representation of the UV/vis Spectrum of protected mercaptodextran substituted with DNP haptens.
  • the number of DNP haptens can be determined from the absorbance at 360 nm and the number of mercapto groups from the increase in absorbance at 343 nm when DTT is added;
  • Figure 12 illustrates the step of deprotection of haptenylated mercaptodextran molecule with DTT (before conjugation to nanoparticles the mercaptodextran is purified by gel-exclusion chromatography);
  • Figure 13 illustrates a schematic representation of the self-assembly of haptenylated mercaptodextrans to gold nanoparticles;
  • Figure 14 is a graphical representation of the UN/vis spectra showing the effect of conjugating different numbers of haptenylated mercaptodextrans to gold nanoparticles.
  • the numbers on the spectra correspond to the number of dextran molecules per particle.
  • the particles flocculate on addition of PBS as indicated by the decrease in absorbance at 520 nm;
  • Figure 15 illustrates a schematic, s nm ⁇ ry of a paramagnetic microbead immunoassay for D ⁇ P-gold nanoparticle conjugates
  • Figure 16 is a graphical representation of the UN/vis spectra showing the .absorbance changes that occur when different amounts of antibodies bound to paramagnetic microbeads are rotated with a nanoparticle conjugate solution;
  • Figure 17 is a graphical representation of the absorbance at 520 nm for a gold nanoparticle conjugate solution against the amount of antibodies bound to paramagnetic microbeads added and removed by magnetic precipitation;
  • Figure 19 is a UN/vis spectra of PDP dextrans substituted with different numbers of D ⁇ P haptens.
  • the mean number of haptens per molecule of dextran in order of decreasing absorbance at 360 nm (D ⁇ P) is: 12,8.6, 5.9, 4.4 and 1.2;
  • Figure 20 is a multiwell plate image showing how the amount of GNP conjugate bound to microbeads increases as the number of probe molecules per particle increases. The amount of conjugate bound is proportional to the colour density of the wells;
  • Figure 21 is a graph showing how the colour density of the microbeads in Figure 20 increases as the number of probe molecules per particle increases. Key to relative conjugate concentration: 0.35 (solid line); 0.7 (dashed line); 1.4 (chain dot line). Note that on the greyscale of 0 - 255 high colour density corresponds to low numerical values;
  • Figure 22 is a multiwell plate image showing the results of a reagent limited (competitive) immunoassay for DNP;
  • Figure 23 is a graph showing how the colour density of the microbeads in Figure 22 increases as the amount of DNP decreases. Note that on the greyscale of 0 - 255 high colour density corresponds to low numerical values;
  • Figure 24 shows a UN/vis spectra of 10 nm gold nanoparticles stabilized with different amounts of 70 kDa PDP dextran.
  • the numbers of dextran molecules per particle, in order of increasing absorbance, are 0, 4.3, 4.9, 5.2, 5.1 and 7.1;
  • Figure 25 is an image of 10 nm gold nanoparticles conjugated to different amounts of 70 kDa PDP dextran. The numbers indicate the ratio of dextran molecules to particles;
  • Figure 26 is a graph showing how the maximum absorbance of gold nanoparticles varies for different ratios of 70kDa PDP dextran per nanoparticle. The minimum amount required to prevent any flocculation is taken as the lowest ratio that does not lead to a decrease in the maximum absorbance;
  • Figure 27 is a UN/vis spectrum of 70 kDa PDP dextran substituted with 19-mer oligonucleotides. This spectrum was recorded using a quartz cuvette with a 3mm path length;
  • Figure 28 illustrates the structure of 70kDa PDP dextran substituted with 19-mer oligonucleotides
  • Figure 29 is an image of a multiwell plate showing ohgonucleotide G ⁇ P conjugate hybridised to different amounts of complementary oligonucleotides bound to polymer microbeads; amounts of the latter are given in picomols. This image was acquired with an ordinary document scanner;
  • Figure 30 shows a UN/vis spectrum of 2000 kDa PDP dextran substituted with biotin caproic acid
  • Figure 31 illustrates the proposed structure of PDP dextran substituted with biotin caproic acid
  • Figure 32 is an image of a multiwell plate showing different amounts of biotin conjugate bound to streptavidin-coated microbeads.
  • the control image shows a multiwell plate containing beads that were incubated with the same amounts of conjugate that did not have biotin as the probe molecule. This image was acquired with an ordinary document scanner;
  • Figure 33 is a graph of colour density against the amount of G ⁇ Ps bound to the microbeads in Figure 32.
  • biotin conjugate conjugate without biotin
  • Figure 34 shows a UN/vis spectrum of antibody PDP dextran. This spectrum was recorded using a quartz cuvette with a 3mm path length;
  • Figure 35 illustrates the proposed structure of the antibody linked PDP dextran
  • Figure 36 shows the results of reagent reagent-limited immunoassays for D ⁇ P with antibody conjugate on lateral flow devices
  • Figure 37 shows the proposed structure of haptenylated mercapto dextran used to prepare QD conjugates.
  • Figure 38 is an image showing the results of a reagent limited (competitive) immunoassay for D ⁇ P with QD D ⁇ P conjugate.
  • Figures 1 to 9 have already been described and discussed with reference to the prior art. h the following discussion, reference is made, for convenience, to nanoparticle conjugates wherein the flexible hydrophilic polymer is a dextran or a dextran derivative and wherein the plurality of conjugation substituents capable of binding to nanoparticles are mercapto substituents, or protected substituents which provide, on deprotection, the same type of conjugation as would be provided by mercapto substituents.
  • the flexible hydrophilic polymer and other types of conjugation substituent are contemplated herein and are within the scope of the invention and the ensuing discussion.
  • the conjugation methods described here can be divided into three main steps ( Figure 10).
  • a functional molecule that can participate in biomolecular binding reactions is activated.
  • the activated molecule, and a molecule with a disulphide bond are substituted into dextran under conditions that allow the number and type of substituents to be accurately determined.
  • the substituted dextran is conjugated to the NPs by a process of self-assembly, in which the sulphur atoms of the mercapto groups become chemically bound to the particles.
  • the third step is carried out in such a way that the number of substituted dextrans conjugated to each NP is controlled by the relationship between the size (molecular weight) of the dextran and the diameter of the particle.
  • the invention provides a method (sumijjarized in Figure 10) for synthesizing a surface polymer with a known and controlled number of functional molecules at high concentration prior to conjugation.
  • This surface polymer is conjugated to the nanoparticles by a process of self-assembly in which the sulphur atoms in a plurality of mercapto groups become chemically bound to the nanoparticles.
  • the number of polymer molecules conjugated to each nanoparticle is determined by the size of the polymer and the size of the nanoparticle.
  • nanoparticle conjugates in which a polymer with a known number of functional molecules is conjugated to particles by a plurality of electrostatic, hydrophobic or chemical interactions, such that the number of polymer molecules per particle is determined by the size of the polymer and size of the particle.
  • One advantage of nanoparticles is that they have dimensions that are similar to the molecules that are used in biomolecular assays as shown in Table I: TABLE I
  • Antibody (Immunoglobulin G) 4
  • the invention provides for a method of conjugating a known number of molecules that can participate in biomolecular and other applications to nanoparticles.
  • the molecules are covalently attached to a polysaccharide that is also substituted with a plurality of pendant mercapto groups.
  • the polysaccharide is conjugated to the nanoparticles by a multivalent method in which the sulphur atoms of the mercapto groups are chemically bound to the nanoparticles.
  • the invention provides in one of its aspects a three-step method for preparing nanoparticle conjugates. In the first step of the method a functional molecule is activated.
  • a polysaccharide is substituted with a predetermined number of two or more molecules, one of which is a functional molecule and one of which has a mercapto group, or a chemical group that can be converted to a mercapto group.
  • the second step is carried out under conditions that allow the number and type of molecules substituted into the polysaccharide to be accurately determined.
  • the substituted polysaccharide is conjugated to the ' ftanoparticles by a process of self- assembly in which the sulphur atoms of the mercapto groups become chemically bound to the particles.
  • the second step may be carried out in such a way that the number of substituted polysaccharides conjugated to each nanoparticle is controlled by the relationship between the size of the polysaccharide and the size of the particle.
  • nanoparticle refers to particles with diameters of preferably less than about 100 nm.
  • the said functional molecules are covalently attached to a flexible hydrophilic dextran polymer that is also substituted with mercapto groups or protected mercapto groups. This part of the method is preferably carried out under conditions that allow the number and type of molecules substituted into the polymer to be accurately determined.
  • the substituted polymer is conjugated to the nanoparticles by a process of self- assembly, in which the sulphur atoms of the mercapto groups are chemically bound to the particles.
  • the third part of the method is carried out in such a way that the number of polymer molecules conjugated to each nanoparticle is controlled by the relationship between dextran size and particle size.
  • the flexible hydrophilic polymer into which molecules are to be substituted is preferably derivatized with primary amines that can be covalently attached to substituent molecules.
  • the polymer is preferably an aminopolysaccharide and preferably an aminodextran, where the word dextran refers to any branched polysaccharide of D-glucose, regardless of the branch point of the repeating unit; i.e., 1 2, 1 3, 1 4, etc.
  • Aminodextrans are dextrans that have been derivatized with primary amine (NH 2 ) groups. Methods for preparing aminodextrans include reductive animation of periodate oxidized dextran (Biosens.
  • aminodextrans are substituted either directly with molecules that can participate in the said applications, or indirectly with molecules possessing chemical groups that can be linked to molecules that can participate in the said applications.
  • the direct substitution of molecules into aminodextrans is preferably accomplished by activating the molecules that are to be substituted, or derivatives thereof, with a reactive group.
  • reactive groups are succinimidyl or sulfosuccinimidyl esters, isothiocyanates and sulfonyl chlorides.
  • activated molecules are succinimidyl 6- (biotinamido)hexanoate, fluorescein-5(6)-carboxamido-caproic acid NHS and atrazine NHS ester (Biosens. Bioelectron, 12 (1997) 277-286).
  • the indirect substitution of molecules into the aminodextrans is accomplished by activating the primary amines with a bifunctional reagent.
  • bifunctional reagents are succinimidyl hydraziniumnicotinate and related aromatic hydrazines, C6- succinimidyl 4-hydrazinonicotinate acetone hydrazone and related aromatic hydrazones, succinimidyl 4-formylbenzoate and related aromatic aldehydes, suberic acid bis (NHS ester), 6-(iodoacetamide;)caproic acid NHS ester, succinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate (SMCC), succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), 3- maleimidobenzoic acid NHS (MBS), ⁇ -maleimidobutyric acid NHS (GMBS), ⁇ - maleimidocaproic acid NHS (EMCS) and ⁇ -maleimidopropionic acid NHS (BMPS).
  • the aminodextrans are also substituted with mercapto (thiol, sulphydryl, -SH) groups, or chemical groups that can be converted to mercapto groups by removal of a protecting group.
  • mercaptodextrans have been prepared previously for use as chelating agents (Acta Pharmacologica. Sinica, 11 (1990) 363-367) and for coating macroscopic gold surfaces with dextrans (Biomaterials, 21 (2000) 957-966 and Analyst, 128 (2003) 480-485).
  • Mercapto groups can be substituted into aminodextrans by reaction with reagents such as 2-iminothiolane.
  • Chemical groups that can be converted to a mercapto groups by removal of a protecting group can be substituted into aminodextrans with reagents such as N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), N-hydroxysuccinimide s-acetylthioacetic acid (SATA) and S-acetylmercaptosuccinic anhydride (SAMSA).
  • SPDP can be deprotected with dithiothreitol (DTT) or tris-(2-carboxyethyl)phosphine (TCEP), and SATA and SAMSA can be deprotected with 50 mM hydroxylamine.
  • SATA and SAMSA unlike SPDP
  • SATA and SAMSA unlike SPDP
  • SATA and SAMSA unlike SPDP
  • bifunctional reagents are described in texts such as The Pierce Handbook and Chemistry Of Protein Conjugation And Cross-Linking [Wong, S.S.] CRC Press, Inc., Boca Raton, FI, (1991).
  • the substitution of molecules into aminodextrans is preferably carried out under conditions that allow the number and type of the said molecules to be determined accurately.
  • the molecules are substituted into the aminodextrans at relatively high concentrations such that the degree of substitution can be accurately determined by simple methods such as UN/vis spectroscopy.
  • the polymer is prepared in the absence of interference from the nanoparticles. Preparation at relatively high concentration also facilitates purification and increases the yield of the substituted dextran product.
  • the substituted mercaptodextrans prepared in the first part of the method are conjugated to the nanoparticles.
  • nanoparticle may refer to a metal, metal oxide or semiconductor particle that is preferably less than about 100 nm in diameter. Conjugation is carried out by a process of self-assembly in which the sulphur atoms of mercapto groups or protected disulphide bonds in the dextran become chemically bound to the nanoparticle. When the dextran is substituted with a protected disulphide bond mercapto groups may be generated by spontaneous fission of the said bond on contact with the nanoparticle (J. Am. Chem.
  • Another advantage of the invention is that the number and type of molecules conjugated to each nanoparticle may be finely controlled.
  • the minimum number of mercaptodextran molecules required to stabilize the nanoparticles depends on the size of the mercaptodextran molecules and the size of nanoparticles.
  • the size of a mercaptodextran molecule determines the extent to which it can cover the surface area of a nanoparticle; size is related to the molecular weight (MW) of the dextran, but other factors such as the amount of polymer branching, the number and type of the functional molecules and the number of mercapto groups are also involved.
  • the number of mercaptodextran molecules that can be accommodated by each nanoparticle is limited to one.
  • the number of functional molecules conjugated to each nanoparticle is equal to the number of functional molecules in one molecule of the mercaptodextran.
  • the nanoparticle can accommodate more than one mercaptodextran molecule the number of molecules conjugated to each particle will be a multiple of the number of functional molecules in one molecule of the mercaptodextran.
  • This Example describes the production of protected mercaptodextrans (PDP dextrans) with a known number of haptens.
  • protected mercaptodextrans with a mean of about one hapten per molecule 6(2,4- dinitrophenylamino)-l-aminohexanoic acid [N-hydroxysuccinimide ester] (DNPAH-NHS) and SPDP were dissolved in dry DMSO to final concentrations of 7.2 mM and 0.116 M respectively. This solution (0.2 ml) was added, dropwise with stirring to 10 mg of aminodextran (MW 70,000; 16.2 primary amines per molecule as determined by the phthalaldehyde method described in: Makromol.
  • Protected mercaptodextrans substituted with reactive derivatives of molecules other than DNP can be synthesized by similar methods, and by extension known amounts of more than two molecules (e.g. DNP, biotin and PDP) can also be substituted into aminodextrans.
  • This Example describes the production of protected mercaptodextrans with a known number of proteins.
  • the protein that is to be conjugated does not contain a suitable mercapto group it is thiolated with 2-imminothiolane, or with SPDP followed by reductive deprotection of the disulphide and gel-exclusion chromatography.
  • the latter is preferred because it minimizes the possibility of disulphide crosslinking and allows the average number of mercapto groups substituted into each protein molecule to be determined from the amount of pyridinedithione chromophore released on reduction with DTT.
  • a mercapto groups into antibodies is carried out by adding 75 ⁇ l of 13.5 mM of 2- imminothiolane [HCl salt] in PBS to a stirred solution of 10 mg of antibody (IgG) in 2 ml of PBS. After slant rotating for 1 hour at room temperature the thiolated antibody is purified by dialysis against PBS, or by gel-exclusion chromatography on Sephadex G-25.
  • Fab' fragments which have a single antigenic binding site are particularly useful in the context of the invention because they allow the number of binding sites per nanoparticle conjugate to be limited to one.
  • Fab' fragments are prepared by proteolytic cleavage of antibodies with pepsin followed by reduction with 2-mercaptoethylamine (J.
  • Biochem. 92(1982)1413-1424 They contain a single mercapto group, which is remote from the single antigen binding site, and therefore it is unnecessary to introduce a mercapto group chemically.
  • the ratio of mercapto groups to maleimide groups in the dextran is determined by reacting the protected mercaptodextran with deprotected fluorescein SAMSA (Molecular Probes, Eugene, Oregon, USA) and reacting the deprotected mercaptodextran with 5,5'- dithiobis-(2-nitrobenzoic acid) (DNTB, Ellman's reagent; Anal. Biochem.; 101 (1980) 442-448).
  • the number of maleimide groups is determined from the fluorescence at 520 nm and the number of mercapto groups is determined from the absorbance of the 410 nm (1.36 x 10 4 M "1 cm "1 ).
  • thiolated protein 5 x the molarity of maleimides in the dextran
  • pH 6.0, 0.1 M phosphate buffer and slant rotated for 30 minutes the dextran protein conjugates are purified on Sepharose 4B.
  • Protected mercaptodextrans with a known number of oligonucleotides are prepared by similar methods except that oligonucleotides with a terminal mercapto group are used instead of thiolated proteins.
  • This Example describes the production of gold nanoparticle conjugates with a known number of haptens.
  • the process of self-assembly used to conjugate substituted mercaptodextrans to nanoparticles is shown schematically in Figure 13.
  • Protected mercaptodextrans (PDP dextrans are deprotected by reduction with DTT followed by gel filtration on Sephadex G-25.
  • a known amount of nanoparticles is mixed with different amounts of deprotected mercaptodextran 'dissolved in water and then phosphate buffered sodium chloride is added to give a final concentration equivalent to PBS (15 mM Na Phosphate, 0.15 M NaCl).
  • the minimum number of haptenylated mercaptodextrans required to stabilize the particles depends on the diameter of the particles (see Example 4). Because the number of haptens per molecule of mercaptodextran molecule and number of mercaptodextrans per particle are known the number of haptens per nanoparticle conjugate can be calculated, h the example given here there is a mean of one hapten per mercaptodextran molecule and four mercaptodextran molecules per nanoparticle. Therefore the number of haptens per nanoparticle conjugate is four.
  • the number of mercaptodextrans per nanoparticle, and hence the number of haptens per nanoparticle conjugate can be varied in such a way that a known number of molecules can be conjugated to each nanoparticle.
  • This Example describes an experiment conducted to assess the relationship between the number of dextrans and nanoparticle diameter.
  • the PDP dextrans protected mercaptodextrans
  • SPDP no DNPAH-NHS
  • the aminodextrans into which PDPwas substituted had MWs of 10, 40 and 7 ⁇ ;ld- ) a (Molecular Probes) and 170, 500 and 2000 kDa (Helix Research). They were titrated against gold nanoparticles (GNPs) of known size and concentration (BBInternational, Cambridge, UK) to find the minimum amounts required to prevent flocculation by PBS.
  • GNPs gold nanoparticles
  • Figure 18 shows that there is a linear relationship between the minimum numbers of PDP dextran molecules required to prevent flocculation and the square of the particle diameter. It suggests that if the numbers of functional molecules linked to a molecule of dextran prior to conjugation are known, then the numbers of functional molecules per particle can be calculated.
  • This Example describes the use of mercaptodextran nanoparticle conjugates in biomolecular assays.
  • the biomolecular reaction of haptenylated nanoparticle conjugates to antibody-coated paramagnetic microbeads is summarised in Figure 15.
  • DNP-gold-nanoparticle conjugates prepared as in Example 3 were slow tilt rotated with paramagnetic beads coated with the corresponding antibody (anti-DNP) in PBS-Tween (PBS + 1 mg ml "1 BSA and 0.5% Tween-20). After rotating for ten minutes the beads were magnetically precipitated and the UN/vis spectrum of the supernatant was recorded.
  • Figure 16 shows how the absorbance spectrum changed as the amount of beads increased; no change was observed in control experiments when rotation was carried out in the presence of 10 ⁇ M D ⁇ P, or when beads coated with non-specific (anti-mouse) antibodies were used.
  • ⁇ Ps bound nanoparticles
  • This Example describes an experiment which was conducted in order to modulate conjugate affinity by variation in the number of probe molecules (functional molecules) per nanoparticle.
  • Biotinamidocaproate ⁇ - hydroxysuccinimide ester (Sigma) (0.8 mg) in 25 ⁇ l of DMSO was added to 2 mg of anti-D ⁇ P antibodies (Sigma) in 1 ml of PBS.
  • the solution was slow tilt rotated for 1 hour at room temperature and then purified on Sepahdex G-25.
  • the absorbance of the eluate fraction was 0.894, which corresponds to an antibody concentration of 4.96 ⁇ M.
  • Streptavidin coated beads (0.56 ⁇ diameter; Bangs Laboratories, Fishers, IN) were slow tilt rotated for one hour with biotinylated antibody (50 ⁇ g of antibody per mg beads) and then the beads were separated at 9000 g with PBS-Tween as the wash solution.
  • PDP dextrans substituted with dinitrophenyl (DNP) haptens were synthesized by slow dropwise addition of 125 ⁇ l of 6(2,4-dinitrophenylamino)-l-aminohexanoic acid [N-hydroxysuccinimide ester] (0.25 mg ml "1 in DMSO) to vigorously stirred solutions of the following amounts of 2000 kDa aminodextran (Helix Research) in 1.6 ml of 0.1 M sodium bicarbonate solution: lOOmg, 50mg, 25mg, l ⁇ mg and 5mg.
  • the absorbance peak at 360 nm is due to D ⁇ P and the peaks at 235 and 282 nm are due to PDP; there is a small amount of absorbance at short wavelengths due to dextran. From the dextran concentration and the absorbance at 360 nm (1.74 x 10 4 M “1 cm “1 ) it was calculated that there were 1.2, 4.4, 5.9, 8.6 and 12.0 dinitrophenyl (D ⁇ P) haptens per molecule of 2000 kDa dextran. Titration shows that a minimum of one 2000 kDa PDP dextran molecule per particle is required to prevent the flocculation of 15 nm G ⁇ Ps.
  • the mean numbers of haptens conjugated to 15 nm particles was also 1.2, 4.4, 5.9, 8.6 and 12.0.
  • the same numbers of G ⁇ Ps conjugated to different numbers of haptens were slow tilt rotated with anti-D ⁇ P coated microbeads, for one hour at room temperature, and then the beads were spun down at 300g in PBS-Tween (twice) and at 9000g in water (once).
  • the final pellets were evaporated to dryness in a vacuum centrifuge and resuspended in 25 ⁇ l of water. They were then transferred to an in-house multiwell plate and imaged with a document scanner as shown in Figure 20 This shows that the number of GNPs bound by the antibodies increases as the number of haptens per particle increases.
  • the minimum number of dextran molecules required to prevent flocculation ( ⁇ 8.5 per 10 nm particle in this example) can be determined, as shown in Figure 26.
  • the dextran prevents flocculation non-specific binding to proteins is still possible, and therefore BSA and Tween-20 are added as blocking agents.
  • the K a value for a single dative covalent bond between sulphur and gold has a value of around 1.53 x 10 4 M "1 , which is lower than the K a value of a weak antibody- antigen reaction.
  • Ohgonucleotide beads were prepared by coating white streptavidin beads (0.56 ⁇ diameter; Bangs Laboratories, Fishers, IN) with ohgonucleotide having a 5 '-terminal biotin [5' -ACCGCGTCGCACCTGCCGC- 3'] (Qiagen, Cologne, Germany) in PBS-Tween. Precipitation and washing were carried out with wash solution in a centrifuge at 9000 g.
  • Hydrazide substituted PDP-Dextrans A 25 mg ml "1 solution of C6-Succinimidyl 4-hydrazinonicotinate acetone hydrazone (C6-SANH) (Solulink, San Diego, CA) in dry DMSO was used to prepare a 25 mg ml "1 solution of SPDP-NHS. This solution (0.16 ml) was reacted with 4 ml of 2 mg ml "1 aminodextran (MW 70 kDa: Molecular Probes) in PBS, for 2 hours at room temperature.
  • C6-SANH C6-Succinimidyl 4-hydrazinonicotinate acetone hydrazone
  • the dextran was purified on Sephadex G- 25 with AB (0.1 M sodium acetate, pH 4.5) "as the eluant (70 kDa dextran), and the molarities of PDP and hydrazide groups were determined with DTT, and 4- nitrobenzaldehye respectively.
  • the latter determination was carried out as follows: hydrazide substituted PDP dextran (150 ⁇ g in 150 ⁇ l of MES (: 0.1 M 2-(N-morpholino)ethanesulfonic acid, 0.15 M NaCl, pH 4.7)) was added to 150 MES containing 0.5 mM nitrobenzaldehyde (Solulink, San Diego, CA).
  • Ohgonucleotide Conjugate An excess of ohgonucleotide with a 5' terminal aldehyde group [5'- GCGGCAGGTGCGACGCGGT-3'] (Solulink) was reacted with hydrazide substituted PDP dextran (70 kDa) in AB (0.1 M sodium acetate, pH 4.5), overnight at 4°C.
  • the oligonucleotides are covalently attached to dextran molecules that are also substituted with a plurality of PDP groups.
  • the molar ratio of PDP to hydrazide in the dextran was 4:1.
  • Figure 27 shows the UN/vis spectrum of the dextran after MWCO filtration to remove ⁇ oligonucleotides that were not covalently attached.
  • the structure of the ohgonucleotide substituted PDP dextran is shown in Figure 28.
  • Figure 26 shows an image the conjugate hybridised to microbeads coated with different amounts of target ohgonucleotide; ⁇ 500 femtomoles of the target can be distinguished with the unaided eye.
  • Example 10 This example describes the conjugation of biotin to gold nanoparticles and the use of these conjugates in biomolecular assays.
  • the high affinity interaction between biotin and avidin (or streptavidin) is one of the most widely used methods in bioconjugation chemistry.
  • avidin or streptavidin
  • the conjugation of avidin to GNPs was only reported four years ago.
  • Two different methods for conjugating biotin to GNPs have"been described.
  • One method is similar to previously reported methods for conjugating monothiolated oligonucleotides to GNPs, (J. Phys. Chem.
  • Biotinylated PDP-dextran (2000 kDa) was prepared in the same way as hydrazide substituted PDP dextran in Example 9, except 2000 kDa aminodextran (Helix Research, Springfield, OR) was used instead of 70 kDa aminodextran and biotinamidocaproate N-hydroxysuccinimide ester (Sigma) was used instead of C6-SANH. Biotin was determined with HABA (Biochem. J. 94 (1965) 23c-24c), and PDP with DTT. The minimum amount of biotinylated PDP dextran (found by titration) that prevented flocculation in the presence of PBS, was conjugated to 15 nm GNPs.
  • FIG. 30 shows the UN/vis spectrum of biotin substituted PDP dextran after dialysis. The peaks at 234 and 280 nm are due to PDP; biotin does not make a significant contribution to the spectrum and therefore it was determined by 'displacement of HABA from avidin.
  • the structure of the biotinylated PDP dextran is shown in Figure 31. A minimum of about one dextran molecule per particle is required to prevent flocculation of 15 •nm gold ⁇ Ps and therefore there was a mean 14.6 biotins per particle.
  • Figure 32 shows an image of the beads after rotation with a) biotin conjugate and b) mercaptodextran conjugate.
  • the colour density of the spots was determined after converting to a grey scale and plotted on the graph shown in Figure 33.
  • a graph of colloidal gold in solution is also a curve, even though a graph of absorbance against concentration is linear, indicating that the curvature of the plot is due to the imaging system rather than saturation of the beads by the conjugate.
  • This example describes the conjugation of antibodies to gold nanoparticles and the use of these conjugates in biomolecular assays.
  • the conjugation of antibodies to G ⁇ Ps is usually carried out by a non-specific procedure that is believed to involve ionic attraction between the negatively charged gold and the positively charged protein, hydrophobic attraction between the protein and the gold surface, and dative covalent bonding between the gold and sulphur atoms within the structure of the protein (Bioconjugate Techniques, Academic Press (1996) pp 593-604).
  • the optimum pH for conjugation is close to the isoelectric point (pKj) of the antibodies, but different antibodies have different pKj values and each one must be determined separately.
  • Polyclonal antibodies have a range of pKj values, and therefore the antibodies that are actually conjugated reflect the conditions rather than the range of antibodies "in the original solution. Even monoclonal antibodies may be difficult to conjugate, with mouse subclass IgG3 being particularly challenging.
  • a further drawback of non-specific adsorption is that antibodies are known to dissociate from GNPs (Immuno-gold Labeling In Cell Biology, CRC Press, Boca Raton, FI, (1989) pp. 49-60 and Histochem. •Cytochem. 39 (1991) 37-39) which leads to a decrease in sensitivity. Recently it has been suggested that covalent attachment of antibodies to GNPs may be necessary to harness the full potential of these labels (Anal. Chem.
  • the problems of non-specific adsorption are avoided by using a covalent method.
  • First the antibodies are covalently linked to PDP dextran by stable hydrazone bonds, and then the dextran is conjugated to the NPs by a process of self-assembly, in which a plurality of sulphur atoms form dative covalent bonds with the gold.
  • Antibody Conjugate A 5 fold molar excess of succinimidyl 4-formylbenzoate (SFB) (Solulink) in 25 ⁇ l of DMSO was reacted with 2 mg of anti-DNP (Sigma) in 1 ml of PBS, for 2 hours at room temperature.
  • the aldehyde-substituted antibody was purified on Sephadex G-25 with ABS (15 mM sodium acetate, 0.15 M NaCl, pH 5.5) as the eluant.
  • Reagent limited immunoassays were carried out on lateral flow devices comprising a reagent band of D ⁇ P conjugated to a carrier protein, and a reagent band of antibody binding protein, both immobilised on a porous strip. The ends of the strips nearest the D ⁇ P bands were immersed in 50 ⁇ l aliquots of antibody conjugate containing different concentrations of D ⁇ P. Immersion was continued ( ⁇ 5 minutes) until all of the conjugate had migrated through both reagent bands, and then the strips were dried and imaged with a document scanner.
  • the ratio of PDP to hydrazide in the 2000 kDa dextran was 2.9:1, and there was a mean of 1.4 aldehydes per molecule of antibody.
  • Figure 34 shows the UV/vis spectrum of antibody substituted PDP dextran after MWCO filtration to remove antibodies that were not linked to the dextran.
  • the antibodies and the PDP both contribute to the peak at 280 nm, but separate determination of PDP showed that 63.9% of the absorbance at this wavelength was due to the antibodies.
  • the molecular ratio of PDP to antibody in the dextran was 17.1:1, which shows that many of the hydrazides did not react with antibodies.
  • FIG. 35 The structure of the antibody substituted PDP dextran is shown in Figure 35.
  • the minimum amount of dextran required to prevent flocculation of 30 nm GNPs corresponded to a final antibody concent ation of 7.3 nM. Therefore there were about 26 antibodies per particle.
  • Figure 36 shows the results of a series of reagent-limited immunoassays for DNP carriej-i. : out on lateral flow devices; ⁇ 50 ppb of DNP can be distinguished with the unaided eye.
  • QD quantum dot
  • Small isolated particles ca 2-10 nm
  • ZnS, ZnSe, CdS, CdSe, PbS compound semiconductors
  • QDs quantum dots
  • QDs Compared with conventional organic fluorophores QDs have a number of advantages: many colours can be excited at the same wavelength; size- tuneable emission spectra; widely separated excitation and emission spectra; resistance to photobleaching; and long decay times compared with organic dyes such as fluorescein. The latter two properties in particular make them attractive alternatives to organic dyes as labels for biomolecular assays and cellular imaging. Before they can be used as labels, however, they must be conjugated to a suitable probe molecule. Most QDs are synthesized with an outer layer of some hydrophobic ligand such as TOPO (trioctylphosphine oxide) (J. Am. Chem. Soc 115 (1993) 8706-8715 and J. Phys.
  • TOPO trioctylphosphine oxide
  • Anti-DNP coated paramagnetic beads were prepared as described in Example except that the beads (2.8 ⁇ m; Dynal-(UK) Ltd., Bromborough, Wirral, UK) were precipitated and washed magnetically with an MPC-S sample concentrator (Dynal).
  • Haptenylated mercaptodextran was synthesized by adding '50 ⁇ l of 20 mM 6(2,4-dinitrophenylamino)-l-aminohexanoic acid in DMF to 5 ml of 170 kDa amino dextran (Helix Research) at a concentration of 2mg ml "1 in 50 mM bicarbonate solution.

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Abstract

L'invention concerne un procédé de préparation de conjugats de nanoparticules comprenant les étapes suivantes : protocoles de synthétisation de molécules de produits intermédiaires par introduction d'une pluralité connue de deux ou plusieurs substituants dans un polymère hydrophile souple, l'un des substituants étant capable, éventuellement après déprotection, de se lier à une nanoparticule, les autres substituants étant capables de participer à des applications d'analyse ou autres ; protocoles de mise en contact des molécules de produits intermédiaires avec des nanoparticules pendant une durée déterminée et dans des conditions efficaces pour permettre la liaison d'un nombre connu de molécules de produits intermédiaires avec chaque nanoparticule, en vue d'obtenir le conjugat de nanoparticules.
PCT/GB2003/005157 2002-11-28 2003-11-28 Conjugats de nanoparticules et leur procede de production WO2004047870A1 (fr)

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US9442065B2 (en) 2014-09-29 2016-09-13 Zyomed Corp. Systems and methods for synthesis of zyotons for use in collision computing for noninvasive blood glucose and other measurements
US9554738B1 (en) 2016-03-30 2017-01-31 Zyomed Corp. Spectroscopic tomography systems and methods for noninvasive detection and measurement of analytes using collision computing
US10837018B2 (en) 2013-07-25 2020-11-17 Exicure, Inc. Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
US11123294B2 (en) 2014-06-04 2021-09-21 Exicure Operating Company Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
US11213593B2 (en) 2014-11-21 2022-01-04 Northwestern University Sequence-specific cellular uptake of spherical nucleic acid nanoparticle conjugates
US11364304B2 (en) 2016-08-25 2022-06-21 Northwestern University Crosslinked micellar spherical nucleic acids
US11433131B2 (en) 2017-05-11 2022-09-06 Northwestern University Adoptive cell therapy using spherical nucleic acids (SNAs)
US11633503B2 (en) 2009-01-08 2023-04-25 Northwestern University Delivery of oligonucleotide-functionalized nanoparticles
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EP1696784A4 (fr) * 2003-12-22 2010-10-27 Univ Emory Structures bioconjuguees, procedes de fabrication associes et procedes d'utilisation associes
EP1696784A2 (fr) * 2003-12-22 2006-09-06 Emory University Structures bioconjuguees, procedes de fabrication associes et procedes d'utilisation associes
US11633503B2 (en) 2009-01-08 2023-04-25 Northwestern University Delivery of oligonucleotide-functionalized nanoparticles
US10894963B2 (en) 2013-07-25 2021-01-19 Exicure, Inc. Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
US10837018B2 (en) 2013-07-25 2020-11-17 Exicure, Inc. Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
US11957788B2 (en) 2014-06-04 2024-04-16 Exicure Operating Company Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
US11123294B2 (en) 2014-06-04 2021-09-21 Exicure Operating Company Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
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US9442065B2 (en) 2014-09-29 2016-09-13 Zyomed Corp. Systems and methods for synthesis of zyotons for use in collision computing for noninvasive blood glucose and other measurements
US11213593B2 (en) 2014-11-21 2022-01-04 Northwestern University Sequence-specific cellular uptake of spherical nucleic acid nanoparticle conjugates
US9554738B1 (en) 2016-03-30 2017-01-31 Zyomed Corp. Spectroscopic tomography systems and methods for noninvasive detection and measurement of analytes using collision computing
US11364304B2 (en) 2016-08-25 2022-06-21 Northwestern University Crosslinked micellar spherical nucleic acids
US11696954B2 (en) 2017-04-28 2023-07-11 Exicure Operating Company Synthesis of spherical nucleic acids using lipophilic moieties
US11433131B2 (en) 2017-05-11 2022-09-06 Northwestern University Adoptive cell therapy using spherical nucleic acids (SNAs)

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